Asian Journal of Agricultural Sciences 3(1): 55-57, 2011
ISSN: 2041-3890
© M axwell Scientific Organization, 2011
Received: November 01, 2010
Accepted: December 13, 2010
Published: January 10, 2011
Development of Molecular Markers for Leaf Rust Resistance Genes
Incorporated from Alien Species into Common Wheat
1
N. Shabnam , 1 H. A hmad, 1 G.A . Sahib, 2 S. Ghafoor and 2 I.A. Khan
1
Department of Botany,
2
Department of Gen etics, Hazara University, M ansehra
Abstract: National average yield of wheat in Pakistan is only 2.54 tons per hectare. One of the main reason
for low yield of wheat in Pakistan is leaf rust disease. During present study, RAPD based molecular markers
were developed for three leaf rust resistance genes (Lr47, Lr37 and Lr51) incorp orated to wh eat from wild
relatives. Three sets of near isogenic lines (Anza, Anza+Lr37, Kern, kern+Lr47 , Yecora R ojo and Y ecora
Rojo+Lr51) of wheat were used. A total of 12 RA PD primers were used to identify alien rust resistant genes.
One (OPA 07), three (OPA12, OPA15, OPB11) and two (OPA11, OPB11) R AP D primers were identified which
produced specific bands for alien rust resistant genes Lr47, Lr51 and Lr37, respectively.
Key w ords: Alien gene s, com mon wheat, Lr37, Lr47, Lr51, RAPD , Rust
INTRODUCTION
Common (Bread) w heat (Triticum aestivum L.)
belongs to family gramineae. Genomically it is an
allohexaploid, A A B BD D, having 2n = 6x-42
chromosomes. Planned scientific breeding of wheat has
resulted in the production of high yielding pure line
varieties. But it eroded genetic variability of land races
(Frankel, 1970; Sears, 1993). Wild relatives of wheat
including Triticum sp eltoides, Triticum ventricosum,
Triticum monococcum, Secale cereal, Hordeum vulgare
etc. are rich sources of a number of valuable genes,
especially those responsible for resistance to a-biotic
(cold, drought and salt) and biotic (fungus and inset pests)
stresses and can be utilized for wheat improvement
(McIntosh et al., 1995 ).
W heat rust disease (caused by fungus Puccinia
triticinaea) is the most important disease of wheat all over
the world including Pakistan. There are 3 types of rust (i)
leaf rust or brown rust (caused by Puccinia re condita) (ii)
stem rust or black rust (caused by Puccinia gram inis and
(iii) stripe rust or yellow rust (caused by Puccinia
striiform is). Rust epidemics can cause con siderable
damage (upto 80%) to wheat yield in Pakistan
(Hussain et al., 1987). Presently wheat breeders and
geneticists are extracting rust resistant genes from wild
relatives of wheat and utilizing them for wheat
impro vem ent. A leaf rust resistance gene Lr47 was
derived from short arm of chromosome 7S of Triticum
Speltoides and translocated onto short arm of
chromosome 7A of wheat (Heulgara et al., 2000).
Similarly a rust resistant gene Lr37 has been introgreessed
into common wheat from short arm of chromosome 2N of
Triticum ventricosum (Helguera et al., 2003). Another
leaf rust resistance gene Lr51 has been transferred
from
Triticum
speltoides
to common wheat
(Helguera et al., 2005). During present study RAPD
based molecular markers were developed for alien rust
resistance genes incorporated in wheat from T. speltoides
(Lr47, Lr5 1) and T. ventricosum (Lr37).
MATERIALS AND METHODS
Three set of ne ar isogenic line s (with and w ithout
alien gene responsible for rust resistance) of common
wheat were obtained from Professor Jorge Dubcovsky,
Department of Agronomy and Range Scien ce, U niversity
of California, Davis, USA (Chicaiza et al., 2006).
1 = Yecroa Rojo, 2 = Yecra Rojo + Lr51, 3 = Anza,
4 = Anza+Lr37, 5 = Kern and 6 = Kern+Lr47
Small scale DN A isolation protocol described by Weining
and Langridge (1991) was use d to isolate total genom ic
DNA from young and fresh leaves of the plants. Plants
were grown in pots at department of Botany, Ha zara
University, Mansehra during 2008. About 14 days after
germ ination, 0.5 g fresh leaves were collected in 1.5 mL
eppendorf tubes which w ere placed im med iately in liquid
Nitrogen. Leaf material was crushed with a knitting
needle to a fine powder. Five hundred micro liter DNA
extraction buffer was added and mixed well. Five hundred
:L of Phenol:Chlorofom:Isoamyalcohol (in ratio of
25:24:1) was ad ded and vortex until hom ogeno us mixture
was obtained. The tubes were then centrifuged at 5000
rpm for 5 min. Aqueous phase were transferred to a fresh
Corresponding Author: I.A. Khan, Department of Genetics, University of Karachi, Karachi, Pakistan
55
Asian J. Agric. Sci., 3(1): 55-57, 2011
Fig. 1: PCR amplification profile of near isogenic lines of wheat [(Kern+Lr47 (lane 1) and Kern (lane 2)] using RAPD primer OPD3. Arrow indicates Lr47 specific alleles
tube and 50 :L 3M Sodium A cetate (pH = 4.8) and 500
:L Isopropanol was added and mix gently. Tubes w ere
centrifuged at 5000 rpm for 5 min to make the DNA
pallet. Supernatant was discarded and DNA pallet was
dissolved in 40 :L TE (Tris-EDT A bu ffer). Qu ality and
quantity of DNA was checked on 1% Agarose/TBE gel.
Gels were stained with Ethedium Bromide and visualized
under UV light using “Uvitech” gel documentation
system. Polymerase Chain Reaction (PCR) was carried
out using standard technique (D evos and Gale, 1992).
Seventeen Randomly Amplified Polymorphic DNA
primers viz; OP -A2, -A3, -A6, -A7, -A9, -A11, -A 12, A14, -A15, -B1 1, -C3 and -D 3 (obtained from Operon
Technologies, USA) were used during present study.
susceptible parent “Kern” and can be used reliably as
marker for the presence of leaf rust resistance gene Lr47
incorporated to short arm of chromosome 7A of common
wheat from short arm of chrom osome 7S of Triticum
speltoides.
One (OPA07), three (OPA12, OPA15, OPB 11) and
two (OPA 11, OP B11) RA PD p rimers we re identified
which produced specific bans for alien rust resistant genes
Lr47, Lr51 and Lr37, respectively. While 4 RAPD
primers (OPA07, OPD 03, OPA09 and OPC 03) produced
wheat homoeologous chromosomes specific bands. These
wheat specific primers (OPA07 and OPD 03 for
chromosome 7A, OPA09 and O PC03 for chromosome 2A
of whe at) cannot be used for identification of alien rust
resistant gene s in wheat background but can be used for
mapping wheat homo eologou s chromosomes. For 3
RAPD primers OPA06 and OPA 02 no useful
polymorphism was detected in the sets of ne ar isogenic
lines used during present study.
Present findings further strengthened previous reports
(Khan, 2000; Khan and Shapherd, 1995; Autrique et al.,
1995, Heulgara et al., 2000, 2003, 2005) where various
PCR based markers (RAPDs, ASA, CAPS etc.) were used
to map rust resistant genes incorporated from alien
species into w heat chrom osom es.
RESULTS AND DISCUSSION
During present research, Randomly Amplified
Polymorphic DNA (RAPD) primers we re used to tag rust
resistance genes incorporated from alien species (Triticum
speltoides and Triticum ventricosum) into homoeologous
chromosomes of common wheat (T. aestivum L.). A total
of 12 RAPD primers (OPA02, OPA03, OPA 06, OPA07,
OPA09, OPA11, OPA 12, O PA 14, O PA 15, O PB11,
OPC03, OPD03) were used to identify rust resistant genes
introgressed in common wheat. An example of PCR
amplification of near isogenic lines Kern and Kern+Lr47
using RAPD primer OPA-7 is presented in Fig. 1. A very
strong diagnostic ba nd of appro xima tely 1000 bp was
amplified in the near Isogenic line Kern+Lr47 (indicated
by an arrow in Fig. 1. This band was absent in the
CONCLUSION
The RAP D markers developed during present study
will also be useful in future to reduce the translocated
alien segm ent in w heat background. In this regards it is
56
Asian J. Agric. Sci., 3(1): 55-57, 2011
also suggested that more work should be done to identify
more molecular markers for the resistant genes, so that
more precise shortening of alien segments can be carried
out. This shortening of alien segment can be done by
using second round of ph1b induced homoeologous
recombination and presently developed RAP D markers.
Helguera, M., L. Vanze tti, M. Soria, I.A. Khan, J. Kolmer
and J.D. Dubcovsky. 2005 . Markers for triticum
speltoides leaf rust resistance gene Lr51 and their use
to develop nearisogenic lines. Crop Sci., 45: 728-734.
Heulgara, M ., I.A. Khan and J.D. Dubcovsky, 2000.
Development of PCR marker for wheat leaf rust
resistance gene Lr47. Theor. Appl. G enet., 100:
1137-1143.
Hussain, M., S.F. H assan and M . Kirm ani, 1987.
Virulences in Puccinia re condita in Pakistan during
1978 and 1 979. Proce eding s of the Fifth European
and Mediterranean Cereal Rusts Conference, Bari,
Italy, pp: 21.
Khan, I.A., 20 00. M olecu lar and agron omic
characterization of wheat-A gropyron intermedium
recombinant chromosomes. Plant Breed., 119(1):
25-30.
Khan, I.A. and K.W. Shapherd, 1995. Patterns of
homologous recombination between chromosomes
7Ai and 7 A T riticeae. U nifying plant genomes;
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Symp. Cambridge, pp: 30.
McIntosh, R.A., C.R. Wellings and R.F. Park, 1995.
W heat rusts: An atlas of resistance genes. CSIRO
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W eining, S. and P. Langridge, 1991. Identification and
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ACKNOWLEDGMENT
Laboratory facilities pro vided by the Departm ent of
Genetics, Hazara University are acknowledged.
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